Applications of adaptive optics in microscopy

ABSTRACT

The use of one or more wavefront modulators in the observation beam path and/or illumination beam path of a microscope provide various advantageous results. Such modulators may be adapted to change the phase and/or the amplitude of light in such a way to carry out displacement and shaping of the focus in the object space and correction of possible aberrations. The possible areas of use include confocal microscopy, laser-assisted microscopy, conventional light microscopy and analytic microscopy.

[0001] U.S. Pat. No. 5,504,575, R. Stafford (1993/96):

[0002] Spectrometer based on spatial light modulator and dispersingelement. Uses fibers and optical switches/flexible mirrors to switch thelight to the detector after passing through the dispersing element.

[0003] EPO 167877, Bille, Heidelberg Instruments (applied for 1985):

[0004] Ophthalmoscope with adaptive mirror.

[0005] Definition of Terminology:

[0006] Definition of “wavefront modulator”:

[0007] Within the meaning of the invention, a device for deliberatelyinfluencing the phase and/or the amplitude of a light wave. Based on areflecting optical element (deformable mirror, electrostatic control, orcontrolled by a piezo array, or as a bimorphic mirror) or a transmittingoptical element (LCD or similar unit). It can be built in a continuousor segmented manner. In particular, the segments can be adapted forcontrolling the respective problem.

[0008] Definition of aberrations in the microscope:

[0009] The aberrations of the microscope objective occurring indefocussed operating mode can basically be categorized as correctable ornot correctable. Causally, the aberrations can be divided intoaberrations caused by the objective, aberrations caused by theadditional imaging optics of the microscope, and, finally, those causedby the preparation itself.

[0010] Controlling the wavefront modulator:

[0011] Controlling the wavefront modulator by a computer withappropriate software. The required correcting variables are eithercalculated beforehand (offline) or are calculated from measuredquantities (online, e.g., through a wavefront sensor or by measuring thepoint brightness in the intermediate image).

GENERAL DESCRIPTION OF THE INVENTION

[0012] The invention relates to the expansion of current microscopes byone or more wavefront modulators in the observation beam path and/orillumination beam path of a microscope. The modulator(s) purposelychange(s) the phase and/or the amplitude of the light in such a way thata displacement and shaping of the focus in the object space and acorrection of possible aberrations is achieved. The possible areas ofuse include confocal microscopy, laser-assisted microscopy, conventionallight microscopy, and analytic microscopy.

[0013] Introduction

[0014] In conventional light microscopy, as well as in laser-assistedmicroscopy, the focus of the objective must be displaced with highprecision along the optical axis as well as laterally. In conventionalmicroscopes, this is carried out by mechanical displacement of theobject stage or objective. In addition, in case of illumination by laserradiation, displacements are also necessary in the object space.Consequently, there is a need for three-dimensional focus control in theobject space.

[0015] Based on the principle of the microscope, these displacements canalso be carried out at the wavefront of the beam path. However, thismanipulation must take place in a pupil plane of the beam path. Axialdisplacement of the focus in the object corresponds to a sphericalchange in the wavefront, lateral displacement of a tilt of thewavefront. Also, aberrations in the beam path can be compensated bychanging the wavefront.

[0016] Applications in Conventional Light Microscopy

[0017] Observation Beam Path:

[0018] In order to achieve an axial displacement of the focus in theobject space without changing the distance from the objective to theobject, the wavefront in the pupil of the objective, or in a planeequivalent to the pupil plane, must be spherically deformed. Suchdeformation can be achieved through a wavefront-phase modulator.Illustrations 1 and 1 a show a schematic imaging beam path of an opticallight microscope with an observed object, an objective, and a tube lensfor generating an intermediate image which can be viewed by eyepieces,not shown. A wavefront modulator, according to the invention, isarranged between the tube lens and objective. The wavefront which iscurved after the objective is corrected by the wavefront modulator bycompensating for the aberrations of the objective.

[0019] Calculations have shown that with radii of curvature of thewavefront in the pupil of between −3.0 m and 1.5 m, the focus can bedisplaced by more than 1.5 mm. This depends on the objective that isused; in the present case, the data refer to the Epiplan-Neofluar20×/0.5. Displacements in the range of several tens of micrometers aresufficient in most cases. As mathematical calculations have furthershown, the interval of a possible focus displacement decreases as themagnification of the objective increases. However, since the objectiveis not calculated or designed for this spherically deformed wavefront inthe entrance pupil, aberrations through the objective during defocussingcannot be prevented.

[0020] A focus displacement of the kind mentioned above withoutmechanical influence of the objective has several advantages. First, anymechanical influencing of the object by the microscope objective iseliminated by the fixed working distance between the front lens of theobjective and the object. Accordingly, it is possible for the first timeto carry out sectionwise image recording with different depth positionsof the observation plane with a static water-immersed object.Previously, a technique of this kind failed as a result of themechanical deformation of the object and its surrounding medium throughmechanical pressure on the preparation.

[0021] The fixed working distance in the microscope also yieldsadvantages in the analytic examination of specimens in the biomedicalfield. When using microtiter plates, a correction of aberrations causedby the microtiter plate can be compensated. The microtiter plate can beincluded optically in the beam path and the microscope objective can bepartially (e.g., the front lens) integrated therein.

[0022]FIG. 1b shows a construction of an optical light microscope withdeformable mirrors which correct the wavefront in the direction of thetube lens. A first modulator arrangement and a second modulatorarrangement are included in the imaging via a beam splitter between theobjective and tube lens. In addition, optics for pupil adaptation areprovided in front of each modulator arrangement. The above-mentionedarrangements will be described more fully in connection withIllustration 7. A correction of aberrations due to the preparation andthe surrounding medium of the specimen is also possible by means of asuitable deformation of the wavefront through the wavefront modulator.This is shown in Illustration 2. The wavefront which is distorted byaberrations is corrected by the wavefront modulator arranged between theobjective and the tube lens. However, the spherical components in thewavefront correction are not sufficient for this purpose; asphericalcomponents must be included. Annular actuators are sufficient forrotationally symmetric aberrations (all terms of higher-order sphericalaberration). For angle-dependent aberrations, segmented actuators mustbe used (FIG. 4). These segmented actuators can either be integratedtogether in the same wavefront modulator or two independent modulatorscan be used in different pupil planes. In the first case, the number ofactuators is in a quadratic scale, in the latter case a linear scale,with the required resolution, which means a reduction in the complexityof control electronics.

[0023] Currently obtainable phase modulators are limited with respect toamplitude and with respect to the maximum phase gradients that can begenerated. This in turn limits the possibilities for correction far awayfrom the working point of the objective. A conceivable alternativeconsists in combining adaptive optics with conventional glass optics.The latter serve to generate a large phase gradient or large wavefrontamplitudes, and precision tuning is achieved by means of adaptiveoptics.

[0024] When displacing to a greater focus distance, the required convexwavefront of the pupil results in a vignetting which leads to lowerlight efficiency and a reduction in usable aperture. This limitation isdesign-related and can be taken into account, in principle, in futureoptical configuration of an objective.

[0025] Further, aberrations occurring in the beam path when the focus isdisplaced can result in distortions of the image. In order to correctthese aberrations, non-spherical components can be superposed on thewavefront as was indicated above. According to mathematicalcalculations, a considerable improvement can be achieved in the image(beam ratio greater than 98%) even with small rotationally symmetriccomponents of orders r⁴ and r⁶ (spherical aberration of higher order) atthe wavefront.

[0026] A further advantage of the process consists in the achromaticbehavior of a reflection-based wavefront modulator. With a suitablecoating of the membrane mirror, the entire spectral range from low UV tofar IR can be phase-modulated. Chromatic aberrations (with the exceptionof absorption effects) are ruled out. This results in new processes forchromatic correction in image generation. For this purpose, theillumination is adjusted sequentially to different wavelengths, whereinthe wavefront modulator is adjusted to the suitable optical correctionfor each of the individual wavelengths. In this way, a set of imageswith optimum chromatic correction is obtained which, when superposed,give a white-light recording of high chromatic correction which cannotbe achieved in the same way through the use of conventional glassoptics. Accordingly, in principle, an objective with a wavefrontmodulator can be corrected in an optimum manner on as many wavelengthsas desired in the optical spectrum.

[0027] The required wavefronts initially have only a rotationallysymmetric character for displacement of the focus and for correction ofspherical aberrations. In order to generate such wavefronts in the pupilof the microscope objective, the adaptive optics must have adistribution of actuators with increasing spatial frequency toward theedge (Illustration 4) because the largest gradient in the wavefrontoccurs at the edge. Illustration 4 shows various actuator structureswith increasing spatial frequency in FIGS. 4a)-4 c) and with segments in4 d), e.g., for correcting astigmatism and coma.

[0028] In camera-assisted image generation, the effect of pixelmismatching occurs especially with high spatial resolution. In thiscase, the microscope image is displaced toward the camera so that theindividual images of the video signal are spatially displaced. Thisproblem can be eliminated by a variable tilt component in the wavefrontof the imaging signal. The unsteady movements of the image signal can beeliminated by regulation and a static image can accordingly be generatedby suitable regulation.

[0029] Another problem in camera-assisted image recording is fieldcurvature. The field curvature can be improved during operation, at theexpense of other parameters such as chromatic correction, through theuse of a wavefront modulator in the imaging beam path.

[0030] Illumination Beam Path:

[0031] A flexible configuration of optics, improved opticalcharacteristics of the microscope, and new illumination techniques canbe realized in the illumination beam path by introducing adaptiveoptics. In a similar way to the observation beam path, a wavefront phasemodulator can optimize the imaging of the illumination burner (or of thelaser, as the case may be) in the object plane. Likewise, in the case ofcritical illumination, an even illumination of the object space can beadjusted. Illustration 3 shows a wavefront modulator between thecollector and condenser which are arranged following an illuminationburner.

[0032] The illumination intensity in the object plane can be optimizedspatially with respect to intensity and homogeneity by a wavefrontamplitude modulator. In principle, a manipulation of the pupil ispossible in this way. An oblique illumination of the object space can beachieved by specifically changing the tilt component or tilt proportionof the wavefront.

[0033] Applications in Confocal Microscopy and LSM

[0034] By using laser light for illumination, the applications can berealized in confocal microscopy more readily than in conventional lightmicroscopy.

[0035] Illumination

[0036] When using a laser for illumination, the use of a wavefrontmodulator is advantageous already when coupling into the illuminationfiber. In this respect, it is possible to realize variable adaptationoptics whose focal lengths and imaging scale ratio are adjustable independence on the beam characteristics of the laser(s) and the utilizedfiber(s) in order to achieve an optimum in-coupling into the fiber.Arrangements based on the same principle can also be used in couplingillumination fibers to the microscope optics. Because of the rapidity ofthe modulators, time-resolved measurements and multiplexing procedurescan also be realized in order to switch between one or more lasers anddifferent fibers.

[0037] In confocal imaging, the transmission can be adapted dynamicallythrough the defining pinhole. Both the position and diameter of thefocus are variable within wide limits. The illumination laser, orlasers, can thus be adjusted in an optimum manner based on requirements.Not only rotationally symmetric apertures but also those having otherkinds of outlines or profiles such as lozenge-shaped or rectangularapertures of the type always occurring in pinholes realized in practicecan accordingly be adapted and optimized to maximum transmission orminimum diffraction losses. An optimization of this kind can beinitiated statically by parameters that are calculated beforehand on theone hand or can be regulated during operation to determined optimizingparameters.

[0038] As in conventional light microscopy, the chromatic correction canalso be adjusted in dependence on the utilized illumination laser.Sequential images can be recorded at different wavelengths, with optimumchromatic correction in each instance, through the use of fast,synchronously controlled wavefront modulators in the laser inputcoupling and in the illumination optics and recording optics.

[0039] Realization

[0040] Wavefront modulators are currently obtainable in differentconstructions (Illustration 5), for example, transmitting modulatorsbased on LCD (Illustration 5) or reflecting modulators with movablemembranes. These may be distinguished, in turn, according to their typeof actuating elements: piezo-controlled (5 b), electrostatic (5 a) orbimorphic membranes (5 c). Although the invention is directed generallyto wavefront modulators, the electrostatic membrane mirror is especiallyemphasized in this respect in view of its numerous advantages.

[0041] A micro-fabricated monolithic membrane mirror of the typementioned above, shown in more detail in FIGS. 6a, b with membrane M anddriving electrodes E, is distinguished by excellent flatness and goodoptical quality of the reflecting surface (better than λ/20), smallphysical size (2-20 mm), hysteresis-free control with low voltages (lessthan 100V), high mechanical cutoff frequency of the membrane (severalMHz), large travel or lift (≈100 μm), and therefore small radius ofcurvature (down to 1 m), and an actuator structure that is variablewithin wide limits and has a high spatial density. The minimum actuatorsize is ultimately only limited by the condition that it must be greaterthan the distance between the electrode and the membrane.

[0042] The great advantage of the electrostatic membrane mirror consistsin the fact that only a constant potential need be applied to theactuator electrode for adjusting a parabolic shape. The parabolic shapeof the mirror is given at constant driving of the electrodes by thephysical behavior of the membrane (constant surface force). Accordingly,high dynamics can be achieved in the correcting variable (mirror lift)with low dynamics in the control variable, that is, the applied voltage.

[0043] Illustration 7 shows a laser scanning microscope with ashort-pulse laser, especially for multiphoton excitation. This will beexplained more fully hereinafter.

[0044] Nonlinear Processes:

[0045] In nonlinear processes, the detected signal depends on the nthpower of the excitation intensity. High intensities are necessary forexcitation. These high intensities are achieved through the use of shortpulse lasers and the subsequent diffraction-limited focussing withmicroscope objectives. Therefore, it is the aim of the arrangement torealize the smallest possible (i.e., most ideal) focus and the shortestpossible pulse length in the specimen. In this way, high intensities canbe achieved in the specimen. Nonlinear processes are, for example,multiphoton absorption, surface second harmonic generation (SSHG), andsecond harmonic generation (SHG), time-resolved microscopy, OBIC, LIVA,etc. The invention will be explained more fully in the following withreference to two-photon microscopy.

[0046] WO 91/07651 discloses a two-photon laser scanning microscope withexcitation through laser pulses in the subpicosecond range at excitationwavelengths in the red or infrared region.

[0047] EP 666473A1, WO 95/30166, DE 4414940 A1 describe excitations inthe picosecond range, and beyond, with pulsed or continuous radiation.

[0048] A process for optical excitation of a specimen by means oftwo-photon excitation is described in DE C2 4331570.

[0049] DE 29609850 by the present Applicant describes coupling of theradiation of short-pulse lasers into a microscope beam path vialight-conducting fibers.

[0050] Two-photon Microscopy:

[0051] As is well known, two-photon fluorescence microscopy basicallyopens up the following possibilities in contrast to conventionalsingle-photon fluorescence microscopy:

[0052] 1. Realization of a nonlinear excitation probabilityI_(2 hv)=A·I_(exc) ² with the following advantages:

[0053] three-dimensional discrimination, i.e., depth discrimination,without the use of a confocal aperture

[0054] bleaching out and destruction of cells takes place—if at all—onlyin the focus

[0055] improved signal-to-noise ratio

[0056] use of new detection methods such as non-descanned detection.

[0057] 2. NIR excitation with femtosecond lasers has the followingadvantages for the examination of biological specimens:

[0058] working in the region of the optical window for biologicalpreparations (700-1400 nm) due to low absorption; this method istherefore also suitable for the examination of living preparations

[0059] low loading of cells due to low mean excitation output

[0060] high penetration depths due to low scatter.

[0061] 3. The excitation of so-called UV dyes without the use of UVlight means that no UV optics are necessary.

[0062] 4. In two-photon excitation, there are broad-band excitationspectra of the dyes. Therefore, it is possible to excite very differentdyes with only one excitation wavelength.

[0063] When ultrashort pulses pass through a dispersing medium, e.g.,glass or a preparation, the following effects take place in particular:

[0064] 1. Group Velocity Dispersion (GVD)

[0065] Femtosecond laser pulses have a spectral width of severalnanometers. The red-shifted wavelength components propagate more swiftlythrough a positive-dispersive medium (e.g., glass) that the blue-shiftedwavelength components. There is accordingly a temporal widening of thepulses and thus a reduction in peak output or in the fluorescencesignal.

[0066] A pre-chirping unit (pairs of prisms, gratings or a combinationof the two) is a negative-dispersive medium, that is, blue-shiftedwavelength components propagate faster than red-shifted wavelengthcomponents. The group velocity dispersion can accordingly be compensatedby means of a pre-chirping unit.

[0067] 2. Propagation Time Difference (PTD)

[0068] The glass paths differ over the beam cross section, seeIllustration 4. This causes a spatial enlargement of the focus so thatthere is a reduction in the resolving capability and peak output orfluorescence signal.

[0069] This effect can be compensated by means of a wavefront modulator(adaptive mirror). With a modulator of this kind, the phase andamplitude of the light wave in the excitation beam path can beinfluenced in a directed manner. A reflecting optical element (e.g., adeformable mirror) or a transmitting optical element (e.g., a LCD) arepossible modulators.

[0070] 3. Wavefront distortion through scattering anddiffraction/refraction

[0071] This distortion can be caused by the utilized optics themselvesor by the specimen or preparation. As in the second effect, thewavefront distortion likewise results in deviations from the idealfocus. This effect can also be compensated by a wavefront modulator (see2).

[0072] The effects mentioned above are generally dependent on the depthof penetration into the preparation.

[0073] The object of the arrangement is to compensate for the GVD, PTDand wavefront distortion synchronously as a function of the depth ofpenetration into the preparation in order to achieve short pulse lengthsand the most ideal possible small focus in the preparation even withextensive penetration depth.

[0074] A possible construction of the arrangement is shown inIllustration 7. The radiation of a short pulse laser KPL passes into apre-chirping unit PCU and then travels, via beam splitter ST1 and beamsplitters ST2, ST3, to two adaptive component elements AD1, AD2. Thefirst component AD1 (coarse) is used for coarse adjustment of thewavefront. It is accordingly possible to shift the focus in thez-direction. The wavefront distortions and the PTD effects arecompensated by the second component AD2 (fine). The laser light reachesthe object via beam splitter DBS, x/y-scanning unit, optics SL, TL, andmirror SP and objective OL. The light coming from the object travelsback via beam splitter DBS, lens L, pinhole PH and filter EF to adetector, in this case a PMT, which is connected in turn with a controlunit as are the PCU, AD1 and AD2.

[0075] The adjustment of the adaptive elements AD1, AD2 and thepre-chirping unit, for example, can be effected in this way until amaximum signal is present at the detector PMT. The beam path shown inthe drawing is particularly advantageous for an inverse microscope inwhich observation takes place from below in that the specimen remainsfully accessible for possible manipulation.

[0076] Illustration 6 shows the basic construction of an adaptivemirror. It comprises a highly reflecting membrane (e.g., siliconnitrate) and a structure with electrodes. By specifically controllingthe individual electrodes, the membrane situated above the latter can bedeformed and the phase front of the laser beam can be influenced. Thedeformations of the phase front which occur when the pulses pass throughthe system and through the specimen can accordingly be compensated.

[0077] The pre-chirping unit can comprise one or more prisms or gratingsor a combination thereof. Illustration 8 shows possible arrangementswith prisms (8 a), gratings (8 b), and with prisms and gratings (8 c).The manner of operation is explained more fully in 8 a with reference toa prism compressor. The spectral width of a femtosecond laser pulse isseveral nanometers. When the laser beam passes through the first prism,the beam is broken up into its spectral components. Subsequently, thespectral components travel different glass paths in the second prism.Red-shifted wavelength components are accordingly retarded in relationto the blue-shifted components. The pre-chirping unit accordingly actsas a negative-dispersive medium and a compensation of GVD is alsopossible.

[0078] For the first time, through the use of the arrangement describedabove, the advantages of the excitation of nonlinear processes can beutilized to their full extent and the use of low-power femtosecondlasers is made possible even at greater depths of penetration into thespecimen.

[0079] High peak outputs can accordingly be realized with the use of lowmean excitation outputs so that loading of the biological preparationsor specimens can be kept low, and a high signal-to-noise ratio and highresolution can be achieved in the axial and lateral directions.

[0080] In all of the arrangements described above, the wavefrontadaptation can be advantageously detected and monitored, and adjusted ina defined manner, by means of a wavefront sensor which communicates withthe microscope beam path via a beam splitter (not shown).

1. Arrangement of adaptive optics in the observation beam path of anoptical light microscope subclaims referring to 1 transmitting wavefrontmodulator between objective and tube lens reflecting wavefront modulatorcoupled in between objective and tube lens via beam splitter 2.Wavefront modulator arranged in the illumination beam path of an opticallight microscope subclaims referring to 2 arranged between light sourceand condenser transmitting wavefront modulator
 3. Laser scanningmicroscope with at least one adaptive optical element arranged followingthe laser subclaims referring to 3 first and second adaptive opticalelement for coarse and fine adjustment adaptive optical element isreflecting wavefront modulator laser is short pulse laser pre-chirpingunit provided in addition combination pre-chirping unit and adaptiveoptics in the beam path of a laser scanning microscope with short pulselaser for multiphoton excitation